Analysis on Drug-Resistance-Associated Mutations among Multidrug-Resistant Mycobacterium tuberculosis Isolates in China

As the causative bacteria of tuberculosis, Mycobacterium tuberculosis (M. tb) is aggravated by the emergence of its multidrug-resistant isolates in China. Mutations of six of the most frequently reported resistant genes (rpoB, katG, inhA, embB, gyrA, and rpsL) were detected for rifampicin (RIF), isoniazid (INH), ethambutol (EMB), ofloxacin (OFX), and streptomycin (STR) in this study. The amino acid missense mutations (MMs) and their corresponding single nucleotide polymorphism mutations for all drug-resistant (DR) isolates are described in detail. All isolates were divided into non-extensively drug-resistant (Non-XDR) and preXDR/XDR groups. No statistical differences were detected among MMs and linked MMs (LMs) between the two groups, except for rpsL 88 (p = 0.037). In the preXDR/XDR group, the occurrence of MMs in rpoB, katG, and inhA developed phenotypic resistance and MMs of rpoB 531, katG 315, rpsL 43, and rpsL 88 could develop high levels of DR. It is necessary to carry out epidemiological investigations of DR gene mutations in the local region, and thus provide necessary data to support the design of new technologies for rapid detection of resistant M. tb and the optimization of detection targets.


Introduction
Mycobacterium tuberculosis (M. tb) isolates with multidrug resistance (MDR) are more difficult to treat than drug-susceptible tuberculosis (TB). Close to half a million people developed TB that was resistant to rifampicin worldwide in 2019, and of these, 78% had multidrug-resistant TB (MDR-TB) [1]. The appearance of MDR-TB, including extensively drug-resistant TB (resistance to isoniazid, rifampicin, one fluoroquinolone, and one secondline injectable drug (XDR-TB)) [2], was caused mainly by ineffective treatment, when antibiotics for individual patient are improperly selected or taken, lack of drug-susceptibility testing (DST), and not adopting standard regimens based on the recommendations of the WHO [3,4]. Incorrect diagnosis and treatment of drug-resistant tuberculosis is associated with morbidity, mortality, and ongoing transmission of infection.
As one of the countries with the heaviest burden of MDR-TB, China has reported more and more severe drug-resistant isolates in recent years [1,5]. A baseline survey of drug resistance carried out in 2007 showed that the incidence of multidrug resistance in newlytreated tuberculosis patients and retreated patients was 5.7% and 25.6%, respectively [5].
It is essential to have accurate and rapid diagnosis of MDR-TB, which mainly depends on in vitro testing using either phenotypic methods or molecular techniques [6]. Conventionally, the diagnosis of drug resistance in TB isolates has relied heavily upon culture-based phenotypic DST in liquid or solid media, which has high requirements for TB laboratories, including biosafety conditions, training of technicians, and a long period of weeks to months of incubation. Given the challenges associated with phenotypic DST, rapid molecular tests are increasingly being applied in laboratories, thus providing earlier initiation of appropriate treatment for patients with drug-resistant TB to shorten the regimen.
Drug resistance in the TB complex is mainly conferred through point mutations in specific gene targets, though cell wall structure and the efflux pump also contribute to drug resistance of TB. The reported main amino acid missense mutations (MMs) associated with drug resistance occur in rpoA, rpoB, and rpoC for rifampicin (RIF); katG, inhA, ahpC, and ndh for isoniazid (INH); embB and embC for ethambutol (EMB); rpsL, rrs, gidB, eis, and tlyA for streptomycin (STR); and gyrA and gyrB for fluoroquinolones (FQs) such as ofloxacin (OFX) [7]. For example, 96% to 100% of RIF-resistant M. tb isolates have at least one mutation (the most common mutations are in codons 516, 526, and 531) in the RIF-resistance determining region of rpoB, which encodes the RNA polymerase subunit [8].
Mutation sites of TB drug resistance vary between different countries and regions. A general description of the distribution of these sites is still lacking in China. In this study we analyzed the main mutation sites and frequencies on an amino acid level and base level in order to find the distribution of resistance-associated gene mutations, which will help improve the existing rapid drug-resistance-detection technology and explore new targets for drug activity.

Drug Susceptibility Test
Forty-six clinical isolates of preXDR/XDR and M. tb H37Rv isolate were tested for minimum inhibitory concentration (MIC) of five anti-TB drugs: RIF, INH, EMB, OFX, STR. Mid-log phase mycobacterial cultures in 7H9 medium were prepared and diluted to OD 600 0.02. The diluted bacterial suspension (200 µL) was added to two-fold serially diluted anti-TB drugs (5 µL) in a flat bottom 96-well plate with various concentrations of anti-TB drugs and incubated at 37 • C for one week. Then, 25 µL of 0.02% Resazurin (Sigma) was added to each well and plates were re-incubated for 2 days. A change in color from blue to pink indicated the growth of bacteria. The MIC was defined as the minimum concentration of the anti-TB drugs that prevented the color change. The critical concentrations were 1, 2, 5, 2, and 2 µg/mL for RIF, INH, EMB, OFX, and STR, respectively [15]. To control this effect in this study, a positive control, negative control, and the MIC test for each tested isolate were employed twice in parallel in the study.

DNA Extraction
All the selected clinical isolates were subcultured twice on Löwenstein-Jensen medium from the M. tb stock solutions stored at −70 • C in small aliquots. DNA extraction was carried out by QIAamp DNA mini Kit (QIAGEN, Hilden, Germany). The isolates were removed from the slants of culture medium with an inoculation loop and suspended in 180 µL of Buffer ATL by vigorous stirring. The bacterial pellet was collected after centrifugation for 10 min at 7500 rpm and suspended in 180 µL of the enzyme solution (20 mg/mL lysozyme; 20 mM Tris·HCl, pH 8.0; 2 mM EDTA; 1.2% Triton). The mixture was incubated for 30 min at 37 • C, then added with 20 µL proteinase K and 200 µL buffer AL and mixed by vortexing. After incubation at 56 • C for 30 min, the remaining steps followed the "Protocol: DNA Purification from Tissues" from step 6. Finally, the supernatant was transferred to another 1.5 mL tube and preserved at −20 • C until further use for polymerase chain reaction (PCR).

Drug-Resistance-Related Genes and DNA Sequencing
The six most frequently reported resistant genes detected for RIF, INH, EMB, OFX, and STR were rpoB, katG, inhA, embB, gyrA, and rpsL) were. PCR primers of target genes are listed in Table S1 in Supplementary Materials. The primers (from Sangon Biotech, Beijing, China) and the genomic DNA was used as the template to perform PCR amplification as follows. Each PCR mixture was prepared in a volume of 50 µL containing 25 µL of 2 × PCR mixture, 2 µL of DNA template, and 0.2 µM each primer set. PCR was done under the following conditions: initial denaturation at 95 • C for 5 min and then 35 cycles of denaturation at 94 • C for 30 s, annealing at 65 • C for 30 s, and extension at 72 • C for 30 s, followed by a final extension at 72 • C for 10 min. The PCR products were purified and sequenced by Sangon Biotech (Shanghai, China). Mutations were identified using BLAST and compared with those of M. tb H37Rv isolate using the ClustalW multiple sequence alignment. The impacts of MMs on protein structure were indicated by SMART (Simple Modular Architecture Research Tool) and AlphaFold [16].

Statistical Analysis
The data collected were compiled and analyzed with SPSS statistical software version 24.0 (IBM, New York, NY, USA). The chi-square test or Fisher s exact test was used to compare the two groups of data. When the sample size of the two groups was greater than 40, the chi-square test was performed; when the sample size of one group was less than 40, the fisher exact test was used. The significance threshold was set to 0.05.

Amino Acid Missense Mutation Types and Their Corresponding SNP Mutation Types for All Drug-Resistant Isolates
For all drug-resistant isolates, the most common types of resistance-related genes (rpoB, katG, inhA, embB, gyrA, and rpsL) were analyzed for the incidence of MMs and the corresponding single nucleotide polymorphism mutations (SMs). The main results can be seen in Figure 1. The details can be found in Table S2 in Supplementary Materials. The predicted impacts of MM substitutions for protein function are shown in Figure 2.

Statistical Analysis
The data collected were compiled and analyzed with SPSS statistical software version 24.0 (IBM, New York, NY, USA). The chi-square test or Fisher′s exact test was used to compare the two groups of data. When the sample size of the two groups was greater than 40, the chi-square test was performed; when the sample size of one group was less than 40, the fisher exact test was used. The significance threshold was set to 0.05.

Amino Acid Missense Mutation Types and Their Corresponding SNP Mutation Types for All Drug-Resistant Isolates
For all drug-resistant isolates, the most common types of resistance-related genes (rpoB, katG, inhA, embB, gyrA, and rpsL) were analyzed for the incidence of MMs and the corresponding single nucleotide polymorphism mutations (SMs). The main results can be seen in Figure 1. The details can be found in Table S2 in Supplementary Materials. The predicted impacts of MM substitutions for protein function are shown in Figure 2.

Comparison for the Occurrence of Amino Acid Missense Mutation Types between Non-XDR and preXDR/XDR Groups
By analyzing the differences in the main MM types of the five drugs between the Non-XDR and preXDR/XDR groups, we found that all MM types of the other four drugs were not significantly different except for streptomycin (Figure 3). Among the two MM types of streptomycin, rpsL 43 was not significantly different, and rpsL 88 was significantly different (p < 0.05) between the two groups. The details can be found in Table S3 in Supplementary Materials.

Correspondence between MM Type and MIC Value in preXDR/XDR Isolates
All 46 preXDR/XDR isolates had 27 preXDR and 19 XDR, of which all isolates were resistant to rifampicin and isoniazid, 32 isolates were EMB-resistant, 25 isolates were OFX-resistant, and 33 isolates were STR-resistant ( Table 3). The distribution of MIC values corresponding to different MM types is shown in Figure 4. Antibiotics 2021, 10, x FOR PEER REVIEW 8 of 12 Figure 3. Comparison for the occurrence of amino acid missense mutation types between Non-XDR and preXDR/XDR groups. *, p-value < 0.05 was considered significant.
There were some shortcomings in our research. For the main purpose of this study, which was to screen the DR gene mutation patterns of resistant clinical M. tb, the drugsensitive isolates were not detected, although some MMs were also found in phenotypesensitive isolates [6,8,11,15,26]. The screening of DR isolates only focused on the reported main genes which were significantly related to resistant M. tb, and some new DR gene analysis was not included. Moreover, the isolates included in the study were mainly MDR and XDR, and there was a lack of analysis between different types of resistant clinical isolates, such as single-resistant, MDR, preXDR, and XDR ones. Furthermore, the isolates included in this study spanned more than 10 years, and the drug resistance and susceptibility rates of M. tb for each year were not available.

Conclusions
The effective management of MDR-TB relies on the rapid diagnosis and treatment of drug-resistant infections. Early detection of DR tuberculosis is a key factor in reducing and controlling the spread of these DR strains. It is necessary to carry out epidemiological investigations of DR gene mutations in the region, and provide necessary data support for the design of new technologies for rapid detection of resistant M. tb and the optimization of detection targets.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antibiotics10111367/s1, Table S1: Primers used in this study, Table S2: The incidence of amino acid missense mutation and their corresponding SNP mutation for five drugs in all drug-resistant strains (including Non-XDR and preXDR/XDR), Table S3: The details for the Occurrence of Amino Acid Missense Mutation Types between Non-XDR and preXDR/XDR Groups.